The highly-lethal, pansystemic hemorrhagic fever caused by the mosquito-borne yellow fever virus (YFV) was one of the most feared diseases in Africa, Europe and the Americas, until the live-attenuated 17D vaccine was developed in the 1930's. The natural Asibi isolate of YFV was empirically passaged in primary cultured cells to derive the 17D virus. Genomic sequence comparisons revealed 48 nucleotide and 20 amino acid substitutions that occurred coincident with attenuation of 17D. Although 17D is considered a prototypic live- attenuated virus vaccine, elucidation of the molecular basis for its attenuation and immunogenicity has been greatly impeded by the lack of a small animal disease model. Reasoning that virulence of many pathogenic viruses depends upon evasion and/or antagonism of interferon (IFN)-??? responses by species-specific mechanisms, we investigated the role of IFN-??? in protection of mice from visceroptropic YFV infection. We discovered that wild-type Asibi virus and live-attenuated 17D were readily distinguishable on the basis of their morbidity, mortality and pathogenesis in mice lacking the type I IFN receptor (IFNAR1-/-). In this proposal, this model of YFV disease will be exploited to identify and characterize determinants of YFV attenuation and expose molecular mechanisms that control the virus/host interaction. In Aim 1, we will characterize the pathogenesis of wild-type Asibi and attenuated 17D in IFNAR1-/- mice to pinpoint the step(s) at which 17D replication and dissemination is impeded relative to Asibi. Current models of arbovirus pathogenesis indicate that infection of dendritic cells (DCs) and macrophages in the skin is a crucial early event, in which the viruses exploit migratory properties of activated DCs to effect viremic dissemination. Since Asibi and 17D differ greatly in early viremic potential, we propose that differences in macrophage/DC infection may alter pathogenesis. In Aim 2, we will use chimeric viruses derived from cDNA clones of Asibi and 17D to systematically identify and map the attenuated phenotype of 17D: i) to structural or non-structural gene regions;ii) to specific genes;and finally iii) to single or combinations of nucleotide/amino acid mutations. An extensive panel of chimeric viruses is already available. These studies will begin to elucidate the molecular mechanisms of YFV attenuation and virulence, and will provide a framework for "rational" design of live-attenuated vaccines for protection against other flaviviruses. PUBLIC HEALTH RELEVANCE: The highly-lethal viral hemorrhagic fever caused by the mosquito-borne yellow fever virus (YFV) was one of the most feared diseases in Africa, Europe and the Americas until the live-attenuated 17D vaccine was developed in the 1930's. Even today, over 200,000 West Africans contract YF annually, with tens of thousands of fatalities. The attenuated 17D vaccine strain was derived by repeatedly growing a wild-type YFV isolate (strain Asibi) in cultured cells. Although 17D is considered to be one of the most effective live-attenuated virus vaccines ever developed, the molecular mechanisms that control the attenuation of this live-attenuated vaccine remain a mystery. Our long-term goal is to determine how the host is able to control the 17D infection and elucidate which of the mutations accumulated in the virus genome are responsible for the attenuation. To achieve this goal, Drs. Ryman and Barrett have proposed a consortium agreement in which Dr. Barrett's laboratory will provide viruses to Dr. Ryman's laboratory, where their virulence will be assessed using a newly developed model of YFV pathogenesis and disease. Our understanding of host-pathogen interactions has increased sufficiently to allow rational design of live-attenuated virus strains and the technology exists to introduce and test mutations in genetically-engineered vector systems. It is anticipated that our findings will improve the safety and efficacy of the YFV vaccine, and additionally facilitate the rational design of other live-attenuated virus vaccines, particularly against other pathogenic flaviviruses (e.g., West Nile and dengue viruses) and the closely- related alphaviruses (e.g., eastern equine encephalitis virus), most of which are agents of both emerging infectious disease and bioterrorism/biowarfare.